Acute ischemic stroke diagnosis is an area of high unmet need, with a complex and time-consuming evaluation process prior to administration of tPA and 5-10% of patients who receive intravenous tPA developing significant intracranial bleeding. Circulating biomarkers appear increasingly promising for guiding tPA administration. The well-established connection between time-to-treatment and outcome, however, dictates that any candidate biomarkers used to guide therapy must be measurable in a matter of minutes. [unreadable] [unreadable] This research project will make significant strides toward biomarker-informed prediction of post-tPA intracerebral hemorrhage in ischemic stroke. The research focuses on cellular fibronectin (cFN) and matrix metalloproteinase-9 (MMP-9). Indicators of disruption of vascular integrity, cFN and MMP-9 have been shown in small studies to have significant value for predicting post-tPA intracerebral hemorrhage. ELISA methods for cFN and MMP-9 are well established, but automated point-of-care instrumentation is not currently available. Phase I of this project entails developing a new point-of-care instrument for quantifying cFN and MMP-9 in fingerstick blood samples from acute ischemic stroke patients and validating this instrument both in bench- level experiments and in the clinic. [unreadable] [unreadable] The development process will leverage recent progress in point-of-care instrumentation, including flow- chamber microarrays for multiplexed analyte quantitation with internal controls and cartridge-integrated slit capillary array electroosmotic micropumps for fully active, bidirectional intracartridge sample/reagent transport with a static handheld-cartridge mechanical interface for high reliability. Through an innovative development process, fully functional prototype instruments are scheduled to be ready for clinical evaluation in only nine months. With ELISA-like quantitation performance and time-to-result of less than ten minutes, these instruments will be valuable tools for exhaustively studying cFN and MMP-9 as predictors of intracerebral hemorrhage. Accordingly, a multicenter clinical trial on cFN and MMP-9 making use of the newly developed devices is anticipated for Phase II of the project. [unreadable] [unreadable] This project is pioneering not only in the specific advancement of methods and instrumentation for cFN and MMP-9 quantitation with application to intracerebral hemorrhage, but also by advancing a broad new paradigm for clinical biomarker studies. The analytical methods and instruments developed here are intended to be progenitors of a class of point-of-care devices which can be produced quickly and at low cost to support clinicians' efforts to evaluate new biomarker candidates. The central hypothesis of this project is that integrated, modular point-of-care test devices can be rapidly developed and used to overcome research barriers to biomarker development in time-critical or infrastructure poor settings such as acute stroke. To be most effective, drugs to treat strokes caused by blood clots need to be administered quickly. But because some patients can be harmed, administering clot-busting drugs is not straightforward. This project explores new ways of predicting which patients might be harmed and which are most likely to benefit from stroke therapy, based on the levels of certain markers found in the blood. [unreadable] [unreadable] [unreadable]